Expression of genes of the aflatoxin biosynthetic pathway in Aspergillus flavus isolates from keratitis.

PURPOSE
To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus.


METHODS
The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels.


RESULTS
All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison.


CONCLUSIONS
For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.

Culture conditions: Each fungal strain was first subcultured onto slopes of Sabouraud glucose neopeptone agar (SDA; Hi-Media, Mumbai, India) and incubated at 25-30 °C for 72 h for growth and sporulation. Conidia were harvested in physiologic saline containing 0.04% Tween-80 (Hi-Media) and suspensions of conidia were prepared to contain approximately 1×10 5 colony-forming units (CFU)/ml. One ml of each conidial suspension was then inoculated into 150 ml of sterile glucose-salt medium [16] and incubated at 25-30 °C. The mycelia and the culture filtrates were collected from 7 day-old cultures. The mycelia were frozen and further processed for extraction of RNA, while culture filtrates were screened for presence of aflatoxin.

RT PCR-analysis:
Extraction of total RNA from mycelia-Total RNA was extracted from 100 mg of mycelia by using TRIZOL reagent (Sigma-Aldrich, St Louis, MO). The purity and integrity of the isolated RNA were determined by spectrophotometry and agarose gel electrophoresis.
cDNA synthesis and PCR amplification-Total RNA was used as the template to generate first strand cDNA in a 20 µl reaction volume as follows: 2 µg of total RNA were added to 1 µl of 10 mM dNTPs and 2 µl of 100 µM oligo dTs, made up to 10 µl with RNase-free water, heated at 70 °C for 10 min and added to a reaction mix containing 2 µl of 10× reverse transcriptase buffer, 1 µl of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) enzyme (Promega, Madison, WI) and RNase-free water. The reaction mixture was incubated at 37 °C for 60 min and terminated at 95 °C for 5 min. PCR amplication of the cDNAs of the genes being studied (aflR, aflJ, nor-1, pksA) and of a `housekeeping' gene (β-tubulin [TUB]) was performed with a total reaction volume of 50 µl consisting of PCR buffer (1×), 0.2 mM each of dATP, dGTP, dCTP and dTTP, 0.5 µM of each primer (Table 1) and 1.5 µl of Taq DNA polymerase. After initial denaturation at 95 °C for 15 min, 30 cycles of amplification (denaturation at 95 °C for 30 s, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min) and a final extension at 72 °C for 2 min were performed in a thermocycler (Eppendorf, Hamburg, Germany). The concentration of the template and the number of cycles were optimized to ensure linearity of the response and to avoid saturation of the reaction.
On completion of the PCR reaction, 10 µl of each PCR product were subjected to electrophoresis in a 2% agarose gel containing ethidium bromide. Following electrophoresis, bands corresponding to transcripts of the study genes and the reference gene, TUB, were noted. The gel was photographed using a DS-34 type Polaroid camera and the bands were scanned by an imaging densitometer (Model GS-670; Bio-Rad' Hercules, CA); the intensity of each band was analyzed by Quantity One software (Bio-Rad). The relative expression level of each study gene was calculated as the ratio of the densitometric reading of the study gene transcript to that of TUB. Experiments were performed in replicates.

Assessment of aflatoxin production in culture:
Preparation of culture filtrate-Seven day-old broth cultures of the fungal isolates were successively filtered (Whatman No. 541 and Whatman No. 1 filter paper; Sigma Chemical Co., St. Louis, MO) and then centrifuged at 17,000× g for 30 min at 4 °C in a cooling centrifuge (Heraeus, Hanau, Germany) to yield a supernatant; this was then filtered through a 0.45 μm pore size membrane filter (Millipore, Bangalore, India) to remove any contaminating material, including fungal conidia and bacteria. The culture filtrate thus prepared was screened for the production of aflatoxin.
Screening for aflatoxin production-This was done by thin-layer chromatography (TLC) using a standard method [17] with some modifications. Each culture filtrate was, in succession, extracted with acetone, filtered (Whatman No. 1), extracted with chloroform in a separating funnel for 3 min, filtered, passed through anhydrous sodium sulfate, and concentrated at 60 °C to near dryness. The residue was resuspended in chloroform and spotted in duplicate on 20×20 Quantification of aflatoxin-Aflatoxin detected by the screening process was quantified by the method of Nabney and Nesbitt [18]. The silica gel containing the aflatoxin band was scraped off from the TLC plate and extracted with cold methanol for 3 min. The methanol was then filtered off and the silica gel was washed 5 times with fresh methanol, the combined methanolic filtrate being brought upto 5 ml; the ultraviolet absorption spectrum of the methanolic solution was then recorded. The difference between the optical density of methanolic filtrate at 363 nm and that at 420 nm was determined. This difference was then divided by the extinction coefficient (19,800) of AFB1, and the resulting figure was multiplied by the molecular weight of AFB1 (312) to obtain the concentration of aflatoxin. Statistical analysis: The relative expression levels of the genes studied (levels relative to those of the house-keeping gene TUB) are presented in Table 2 as mean±standard deviation of multiple readings made on 7 day-old fungal cultures. The statistical significance of differences between different groupings of the fungal isolates for each gene studied was determined by one-way ANOVA (ANOVA); where significant differences were noted, intergroup comparisons (between two groups) were performed by post-hoc testing using Tukey's HSD (Statistical Package for Social Sciences-SPSS version 16.0; IBM Corp, Armonk, NY). The results of the statistical analysis for aflR, aflJ, nor-1 and pksA are presented in Appendix 1.
An attempt was also made to determine whether there were significant correlations between the quantum of aflatoxin produced and the relative expression level of each gene (Table 3); this was done by Pearson's rank correlation (Graphpad Instat 3.10; Graphpad Software Inc., San Diego, CA). Figure 1 and Figure 2), detected transcriptional activation (expression) of the aflJ, nor-1, and pksA genes in all four corneal isolates of A. flavus, and expression of aflR in three of the four isolates (Table 2); a 7-day-old culture of C2 did not express aflR, but the transcripts of the aflJ, nor-1, and pksA genes were still detected in this isolate ( Table 2). The mean expression levels of aflR, nor-1, and pksA were significantly higher in the aflatoxigenic corneal isolates (C1, C4) than in the non-aflatoxigenic corneal isolates (C2, C7); the mean expression levels of aflJ in the corneal aflatoxigenic isolates C1 and C4 were significantly higher than that in the non-aflatoxigenic corneal isolate C7 (Appendix 1, a-d). Screening for aflatoxin production by TLC (Figure 3), followed by quantification by spectrophotometry, confirmed the production of AFB1 by corneal isolates C1 and C4, but not by C2 and C7. In the corneal aflatoxigenic isolates, a significant positive correlation was noted between mean aflatoxin production and the mean relative expression level of aflR (p=0.030) only (Table 3).

RT-PCR (
Interestingly, the gene expression results obtained with the four environmental isolates tested as a comparison were mostly similar to those obtained with the corneal isolates ( Figure 1 and Figure 2; Table 2 and Table 3), with some exceptions. In contrast to the corneal isolates, all four environmental isolates were found to express all four genes ( Figure 1 and Figure 2; Table 2). Similar to the corneal isolates, the mean expression levels of aflR, aflJ, nor-1, and pksA were significantly higher in aflatoxigenic environmental isolates (E1, E8) than the levels in non-aflatoxigenic environmental isolates (E2, E9; Appendix 1, a-d). Among the environmental isolates, aflatoxin B1was found to be produced by E1 and E8, but not by E2 and E9 ( Figure 3). Significant (p=0.002 to p=0.035) positive correlations emerged between mean aflatoxin production and mean relative expression levels of all four genes studied in the environmental aflatoxigenic A. flavus isolates (Table 3).

Mean (±SD) relative expression levels** of aflatoxin biosynthetic pathway genes in
There were no significant differences between aflatoxigenic corneal and aflatoxigenic environmental isolates in mean expression levels of all four genes, or between non-aflatoxigenic corneal and non-aflatoxigenic environmental isolates in mean expression levels of nor-1 and pksA (Appendix 1, a-d).

DISCUSSION
The expression of aflatoxin biosynthetic pathway genes has been studied in isolates of A. flavus from the field [13], but not in those from clinical sources. Our investigation is possibly the first to examine the expression of regulatory and structural aflatoxin biosynthesis genes in A. flavus isolates from patients with keratitis.
Accinelli et al. [13] reported that approximately 60% of A. flavus isolates from soil were aflatoxigenic (had the potential to produce aflatoxins). We have previously reported that AFB1 was detected in 80% of growth samples from clinical isolates of A. flavus (from patients with keratitis) but in only 40% of growth samples from environmental A. flavus isolates [14]. We sought to investigate whether the observed differences in aflatoxin-producing ability could be explained by differences in expression of aflatoxin biosynthesis genes between A. flavus isolates from patients with keratitis and those from the environment. We decided to test out this hypothesis by quantifying relative expression levels of the genes in these isolates. We used RT-PCR for this purpose since, although only a semi-quantitative method, it has been found accurate in differentiating between aflatoxigenic and non-aflatoxigenic isolates of A. flavus in agricultural commodities [19], and has also been used by other workers [13,20] to study aflatoxin biosynthetic genes in A. flavus. Thin layer chromatography (TLC) was used to quantify aflatoxin production by the isolates since this is the official method of the Association of Agricultural Chemists (AOAC) and can identify and quantify aflatoxins at levels as low as 1 ng/g. In fact, this method is the basic technology for verification of newer techniques of aflatoxin detection; in other words, this technique can be considered as the 'gold standard'.
Accinelli et al. [13] sought to elucidate the potential of soil A. flavus to produce aflatoxins by detecting, by RT-PCR, transcription of five aflatoxin biosynthesis genes, namely, aflD, aflG, aflP, aflR, and aflS. We selected four genes for study, namely, two regulatory genes, aflR and aflJ, and two structural genes, nor-I and pksA, based on their role in aflatoxin biosynthesis. aflR encodes a putative 47 kDa protein, aflR, which contains a zinc cluster-DNA binding motif that is required for the transcriptional activation of all the characterized aflatoxin pathway genes [21,22]. The specific interaction between aflJ and aflR suggests that aflJ is directly involved in the regulation of transcription of structural genes for enzymes in the aflatoxin pathway [23]. The transcriptional activity of nor-1 and pksA has been found to increase dramatically during aflatoxin accumulation in culture [24][25][26]. In the study by Chang and Hua [27], nonaflatoxigenic isolates were found to be genetically different from an aflatoxin-producing isolate due to the presence of Figure 2. mRNA transcripts of the βtubulin (231 bp), nor-1 (168 bp) and pksA(395 bp) generated by RT-PCR from the corneal and environmental isolates of Aspergillus flavus. Ten µl of each amplified product were loaded on a 2% agarose gel. A: Lanes L1 and L7 were loaded with a 100 bp DNA marker. Lanes L2 to L5 were loaded with RT-PCR products of nor-1, pksA and βtubulin genes from 7-day old cultures of corneal A. flavus isolates C1, C2, C4, and C7, respectively. Lanes L6 and L8 were loaded with controls for the PCR reaction. Lanes L9 to L12 were loaded with RT-PCR products of the nor-1, pksA and β-tubulin genes from 7-day old cultures of environmental A. flavus isolates E1, E4, E8, and E9, respectively.
B: Graphical representation of the relative gene expression levels of the regulatory genes nor-1 and pksA of the corneal and environmental isolates of Aspergillus flavus. polymorphism in pksA, which resulted in the production of a defective polyketide synthase.
In the current study, we observed significantly higher mean (relative) expression levels of aflR in aflatoxigenic isolates of A. flavus (both corneal and environmental) than in non-aflatoxigenic isolates of A. flavus (both corneal and environmental; Appendix 1, a); we also noted a significant positive correlation between expression of aflR and mean aflatoxin production in corneal and environmental aflatoxigenic isolates (Table 3). Interestingly, although aflR was not expressed at all by one non-aflatoxigenic corneal isolate (C2), aflJ, pksA, and nor-1 were still expressed by this isolate; a possible explanation for this is that under normal conditions, expression of structural genes (including nor-1 Figure 3. Thin layer chromatogram of culture filtrates of two corneal and two environmental isolates of Aspergillus flavus. L1: standard aflatoxin B1; L2: Aflatoxin B1 was not detected in the extract of the culture filtrate of corneal isolate C2; L3: Aflatoxin B1 was detected in the extract of the culture filtrate of corneal isolate C1. L4: Aflatoxin B1 was detected in the extract of the culture filtrate of environmental isolate E1. and pksA) requires specific factors (for example, sugars) that induce transcription [22].
Disruption of aflJ in A. flavus has been found to result in failure to produce any aflatoxin pathway metabolites without, however, interfering with the generation of transcripts for many of the aflatoxin structural genes [28]. This latter observation may explain our finding that, although the expression pattern of aflJ differed in some aspects from that of aflR, the expression patterns of nor-1 and pksA still mirrored that of aflR in the isolates of A. flavus.
The results of our study suggest that expression of aflatoxin biosynthetic genes in A. flavus occurs both in strains that produce aflatoxin and those that do not. Possibly, there is a threshold level for expression of the relevant genes. Where genes are expressed above this threshold, aflatoxin production is induced. This would explain our finding of significantly higher expression levels of aflatoxin biosynthetic genes in aflatoxigenic isolates than in non-aflatoxigenic isolates. Interestingly, Kale et al. [29] observed that A. parasiticus variants lacking the sec gene (secstrains) exhibited altered phenotypes and an inability to produce aflatoxin intermediates, compared to sec+ strains; however, the secvariants still produced transcripts of aflatoxin genes, such as aflR, aflD and aflP. We observed significant positive correlations between the expression of all four study genes and mean aflatoxin production in the environmental aflatoxigenic isolates, and between mean expression of aflR and mean aflatoxin production in corneal aflatoxigenic isolates. This suggests that enhanced expression of all four study genes in the environmental aflatoxigenic isolates manifested in increased aflatoxin production whereas enhanced expression of aflR alone in corneal aflatoxigenic isolates translated into increased aflatoxin production. While the exact significance of this finding is uncertain, it is interesting to note that our observation is consistent with those made in studies on A. flavus strains isolated from corn grains collected from fields in Italy [20] and from soils in Canada [13]. In the study by Degola et al. [20] five A. flavus strains expressed all the study genes (aflR,aflS,aflO,aflQ, and aflD), and a good correlation between gene detection, gene expression and aflatoxin production was observed in all these strains except one (aflatoxin non-producing) strain. In the study by Accinelli et al. [13] the overall profile of the genes (aflD, aflG, aflP, aflR, aflS) in the soil, with some genes being expressed and others not expressed, was not generally related to concentration of AFB1; however, the soil in which there was a complete positive gene expression profile also revealed the highest concentration of AFB1 [13]. Thus, the consequences of aflatoxin biosynthetic gene expression in the cornea may differ from that in the environment. strains with that in three progeny nonaflatoxigenic strains, they observed that none of the genes studied were significantly different by their defined parameters; this led them to suggest that loss of aflatoxin production in the progeny strains was not caused directly by altered expression levels of the aflatoxin biosynthetic genes, but, possibly, indirectly by nutritional and physiologic factors that induced changes in primary metabolism, cell cycle and differentiation. Production of aflatoxin by toxigenic Aspergilli is known to be influenced by environmental and nutritional factors, such as temperature, pH, carbon and nitrogen source, stress factors, lipids and certain metallic salts [31].
Aflatoxin is produced by Aspergillus flavus and Aspergillus parasiticus. However, the relevance of aflatoxin as a virulence factor in clinical fungal keratitis is uncertain. The comparatively higher frequency of reports of A.flavus keratitis, compared to the few reports of A.parasiticus keratitis may reflect additional virulence factors in A.flavus such as invasive potential and relative resistance to certain antifungal agents. Although the adaptive value of aflatoxin production is not fully understood, it has been postulated that synthesis of aflatoxins may serve as a defense mechanism against oxidative stress [30]. In this context, it is interesting to note that antioxidants have been shown to reduce aflatoxin production [32] and that a positive correlation has been shown between accumulation of reactive oxygen species and production of aflatoxin by A. parasiticus [33,34]. The results of a series of laboratory experiments suggested that adverse environmental conditions (high temperature, low pH, and nutrient deprivation), but not competition with yeasts and filamentous fungi, helped to maintain aflatoxigenicity over successive generations during serial transfers [35].
A complex interaction of temperature, water activity, incubation period, and substrate has been found to influence the relative concentrations of aflatoxins produced by A. flavus [36]. In this context, the normal corneal temperature of 33 °C to 34 °C [37,38] may stimulate synthesis of aflatoxin. Similarly, a low pH in corneal tissue infected by A. flavus may favor aflatoxin production. Our previous observation [14] that significantly higher percentages of corneal A. flavus isolates than environmental A. flavus isolates were aflatoxigenic may have been because conditions in the cornea possibly influence expression of aflatoxin biosynthesis genes to a greater extent than do conditions in the external (natural) environment.
Thus, corneal tissue infected by A. flavus may provide a favorable setting for increased expression of regulatory (aflR and aflJ) and then structural (nor-I and pksA) aflatoxin biosynthetic genes, therein modulating aflatoxin production by the fungus in the cornea; this may augment the virulence of the fungal isolate infecting the cornea. This effect possibly persists even after the fungus is isolated in culture from corneal scrape material, and subcultured to culture media. These hypotheses need to be confirmed by detection of aflatoxin in corneal tissue infected by aflatoxigenic strains of A. flavus and by demonstrating increased expression of aflatoxin biosynthesis genes and increased production of aflatoxin following transfer of non-aflatoxigenic isolates of A. flavus from the natural environment to corneal tissue.

ACKNOWLEDGMENTS
Instrumentation facility provided by University Grants Commision-Special Assistance Programme (UGC-SAP) of the department of Animal Science, Bharathidasan University is acknowledged. A UGC-SAP Research fellowship to the first author is gratefully acknowledged.